The archaebacteria are a recently discovered group of microorganisms that grow optimally at temperatures above 80.degree. C. Some 20 species of these extremely thermophilic bacteria-like organisms have been isolated, mainly from shallow submarine and deep sea geothermal environments. Most of the archaebacteria are strict anaerobes and depend on the reduction of elemental sulfur for growth.
The archaebacteria include a group of "hyperthermophiles" that grow optimally around 100.degree. C. These are presently represented by three distinct genera, Pyrodictium, Pyrococcus, and Pyrobaculum. Pryodictium brockii (T.sub.opt 105.degree. C.) is an obligate autotroph which obtains energy be reducing S.degree. to H.sub.2 S with H.sub.2, while Pyrobaculum islandicum (T.sub.opt 100.degree. C.) is a faculative heterotroph that uses either organic substrates or H.sub.2 to reduce S.degree.. In contrast, Pyrococcus furiosus (T.sub.opt 100.degree. C.) grows by a fermentative-type metabolism rather than by S.degree. respiration. It is a strict heterotroph that utilizes both simple and complex carbohydrates where only H.sub.2 and CO.sub.2 are the detectable products. The organism reduces elemental sulfur to H.sub.2 S apparently as a form of detoxification since H.sub.2 inhibits growth.
The discovery of microorganisms growing optimally around 100.degree. C. has generated considerable interest in both academic and industrial communities. Both the organisms and their enzymes have the potential to bridge the gap between biochemical catalysis and many industrial chemical conversions. However, knowledge of the metabolism of the hyperthermophilic microorganisms is presently very limited.
The polymerase chain reaction (PCR) is a powerful method for the rapid and exponential amplification of target nucleic acid sequences. PCR has facilitated the development of gene characterization and molecular cloning technologies including the direct sequencing of PCR amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disease disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the exponential amplification of the nucleotide sequence delineated by the flanking amplification primers.
An important modification of the original PCR technique was the substitution of Thermus aquaticus (Taq) DNA polymerase in place of the Klenow fragment of E. coli DNA pol I (Saiki, et al. Science, 230:1350-1354 (1988)). The incorporation of a thermostable DNA polymerase into the PCR protocol obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures which enhance the specificity of primer:template associations. Taq polymerase thus serves to increase the specificity and simplicity of PCR.
Although Taq polymerase is used in the vast majority of PCR performed today, it has a fundamental drawback: purified Taq DNA polymerase enzyme is devoid of 3' to 5' exonuclease activity and thus cannot excise misinserted nucleotides (Tindall, et al., Biochemistry, 29:5226-5231 (1990)). Several independent studies suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis. Reyland et al, J. Biol. Chem., 263:6518-6524, 1988; Kunkel et al, J. Biol. Chem., 261:13610-13616, 1986; Bernad et al, Cell, 58:219-228, 1989. Consistent with these findings, the observed error rate (mutations per nucleotide per cycle) of Taq polymerase is relatively high; estimates range from 2.times.10.sup.-4 during PCR (Saiki et al., Science, 239:487-491 (1988); Keohavaong et al, Proc. Natl. Acad. Sci. USA, 86:9253-9257 (1989)) to 2.times.10.sup.-5 for base substitution errors produced during a single round of DNA synthesis of the lacZ gene (Eckert et al., Nucl. Acids Res., 18:3739-3744 (1990)).
Polymerase induced mutations incurred during PCR increase arithmetically as a function of cycle number. For example, if an average of two mutations occur during one cycle of amplification, 20 mutations will occur after 10 cycles and 40 will occur after 20 cycles. Each mutant and wild type template DNA molecule will be amplified exponentially during PCR and thus a large percentage of the resulting amplification products will contain mutations. Mutations introduced by Taq polymerase during DNA amplification have hindered PCR applications which require high fidelity DNA synthesis.